1. Field of the Invention
This invention relates to affinity chromatography. In one of its more particular aspects, this invention relates to a matrix for use in affinity chromatography and in the immobilization of ligands.
The need for purifying various biologically active materials in a facile manner has long been appreciated. Early methods of enzyme purification, for example, were cumbersome and time consuming. Recently it has been found that biologically active materials can be purified by a process which involves immobilization of the biologically active material, which will be referred to herein as a ligand, upon a suitable polymeric matrix, followed by separation of the immobilized ligand from the mixture in which it is present. The ligand can then be used in its immobilized form, if desired, or it can be released from the matrix on which it is immobilized by suitable chemical treatment and used in its non-immobilized form. The discovery of methods for covalently bonding ligands to polymeric matrices has advanced the practice of enzymology, immunology and various other biological techniques.
2. Prior Art
One of the first methods for immobilizing biological ligands involved treatment of a polymer containing hydroxyl groups with an activating agent such as cyanogen bromide, CNBr. The activated polymer could then be used to directly bind various biological ligands to the polymer by means of covalent bonds. Porath et al. describe several chemical activation methods, including the CNBr method, in Porath, et al., "Immobilized Enzymes", Methods in Enzymology, K. Mosbach, Ed., Vol. 44, pp. 19-45, Academic Press, New York (1976).
Most of the early methods for activating polymers containing hydroxyl groups were subject to certain disadvantages which made their widespread use impractical. In particular, CNBr activation procedures suffer from the following disadvantages: (1) the linkages formed between CNBr-activated hydroxyl containing polymers and the amino groups of ligands which are reacted with the activated polymers are labile; (2) the reaction between the activated polymer and ligand frequently results in the introduction of charged species which interfere with utilization of the reaction product in affinity absorption; and (3) CNBr is a noxious lachrimatory and poisonous chemical which requires special care in its handling.
Efforts to find another method other than the CNBr method for coupling ligands to hydroxyl containing polymers resulted in the use of a number of different reagents including triazine trichloride, N-hydroxy succinimide, 1,1-carbonyldiimidazole and certain epoxy compounds. A method for preparing covalent chromatographic matrices utilizing a hydroxyl containing polymer which has been activated by reaction with 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP) has been described in U.S. Pat. No. 4,582,875, assigned to the same assignee as this invention. However, covalently binding a biologically active substance containing, for example, amino, thiol, or hydroxyaryl groups directly to a polymeric substance may result in steric problems and affect the biological activity of the covalently bound substance.
A spacer, that is, an organic compound, which can be placed between the polymer surface and the ligand, helps to overcome the steric hindrance due to close proximity of the bound ligand to the polymer matrix. Both straight chain spacers and branched chain spacers have been used for this purpose. In P. V. Sundaram, "Potentials of Enzymes Attached to Nylon Tubes in Analysis," Biomedical Applications of Immobilized Enzymes and Proteins, Vol. 2, T. M. S. Chang, Ed., pp. 317-340, Plenum Press, New York (1977), use of a poly-lysine spacer is illustrated (at page 323).
In W. E. Hornby, G. A. Noy and A. B. B. Salleh, "Application of Immobilized Enzymes in Analysis", Biotechnological Applications of Proteins and Enzymes, Z. Bohak and N. Sharon, Ed., pp. 267-278, Academic Press, New York (1977), the use of pectinamine, a polyamine derived from 1,3-diaminopropan-2-ol and pectin, for the covalent attachment of enzymes, is shown (at page 271).
U.S. Pat. No. 4,152,411 describes the preparation and use of a marked spine diagnostic tool, for example, one containing thyroxine, poly-1-lysine and horseradish peroxidase.
The spacer arm concept is discussed in I. Parikh and P. Cuatrecasas, "Affinity Chromatography", Chem. Eng. News, Aug. 26, 1985, pp. 17-32. In this reference, the use of both short alkyl chain spacer arms, such as hexamethylenediamine, and branched copolymers of lysine and alanine as polyfunctional anchoring spacers is disclosed.